The mouse model of laser-induced choroidal neovascularization (CNV) has been used extensively in studies of the exudative form of age-related macular degeneration (AMD). This experimental in vivo model ... [more ▼]

The mouse model of laser-induced choroidal neovascularization (CNV) has been used extensively in studies of the exudative form of age-related macular degeneration (AMD). This experimental in vivo model relies on laser injury to perforate Bruch's membrane, resulting in subretinal blood vessel recruitment from the choroid. By recapitulating the main features of the exudative form of human AMD, this assay has served as the backbone for testing antiangiogenic therapies. This standardized protocol can be applied to transgenic mice and can include treatments with drugs, recombinant proteins, antibodies, adenoviruses and pre-microRNAs to aid in the search for new molecular regulators and the identification of novel targets for innovative treatments. This robust assay requires 7-14 d to complete, depending on the treatment applied and whether immunostaining is performed. This protocol includes details of how to induce CNV, including laser induction, lesion excision, processing and different approaches to quantify neoformed vasculature. [less ▲]

in Neoplasia : An International Journal for Oncology Research (2012), 14(10), 943-51

Carcinoma-associated fibroblasts are key contributors of the tumor microenvironment that regulates carcinoma progression. They consist of a heterogeneous cell population with diverse origins, phenotypes ... [more ▼]

Carcinoma-associated fibroblasts are key contributors of the tumor microenvironment that regulates carcinoma progression. They consist of a heterogeneous cell population with diverse origins, phenotypes, and functions. In the present report, we have explored the contribution of bone marrow (BM)-derived cells to generate different fibroblast subsets that putatively produce the matrix metalloproteinase 13 (MMP13) and affect cancer cell invasion. A murine model of skin carcinoma was applied to mice, irradiated, and engrafted with BM isolated from green fluorescent protein (GFP) transgenic mice. We provide evidence that one third of BM-derived GFP(+) cells infiltrating the tumor expressed the chondroitin sulfate proteoglycan NG2 (pericytic marker) or alpha-smooth muscle actin (alpha-SMA, myofibroblast marker), whereas almost 90% of Thy1(+) fibroblasts were originating from resident GFP-negative cells. MMP13producing cells were exclusively alpha-SMA(+) cells and derived from GFP(+) BM cells. To investigate their impact on tumor invasion, we isolated mesenchymal stem cells (MSCs) from the BM of wild-type and MMP13-deficient mice. Wild-type MSC promoted cancer cell invasion in a spheroid assay, whereas MSCs obtained from MMP13-deficient mice failed to. Our data support the concept of fibroblast subset specialization with BM-derived alpha-SMA(+) cells being the main source of MMP13, a stromal mediator of cancer cell invasion. [less ▲]

In this study, we evaluate the potential involvement of collagenase-3 (MMP13), a matrix metalloproteinase (MMP) family member, in the exudative form of age-related macular degeneration characterized by a ... [more ▼]

In this study, we evaluate the potential involvement of collagenase-3 (MMP13), a matrix metalloproteinase (MMP) family member, in the exudative form of age-related macular degeneration characterized by a neovascularisation into the choroid. RT-PCR analysis revealed that human neovascular membranes issued from patients with AMD expressed high levels of Mmp13. The contribution of MMP13 in choroidal neovascularization (CNV) formation was explored by using a murine model of laser-induced CNV and applying it to wild-type mice (WT) and Mmp13-deficient mice (Mmp13 ( -/- ) mice). Angiogenic and inflammatory reactions were explored by immunohistochemistry. The implication of bone marrow (BM)-derived cells was determined by BM engraftment into irradiated mice and by injecting mesenchymal stem cells (MSC) isolated from WT BM. The deficiency of Mmp13 impaired CNV formation which was fully restored by WT BM engraftment and partially rescued by several injections of WT MSC. The present study sheds light on a novel function of MMP13 during BM-dependent choroidal vascularization and provides evidence for a role for MSC in the pathogenesis of CNV. [less ▲]

The purpose of this study is to explore the function of plasminogen activator inhibitor-1 (PAI-1) during pathological lymphangiogenesis. PAI-1, the main physiological inhibitor of plasminogen activators ... [more ▼]

The purpose of this study is to explore the function of plasminogen activator inhibitor-1 (PAI-1) during pathological lymphangiogenesis. PAI-1, the main physiological inhibitor of plasminogen activators is involved in pathological angiogenesis at least by controlling extracellular proteolysis and by regulating endothelial cell survival and migration. Protease system's role in lymphangiogenesis is unknown yet. Thus, based on its important pro-angiogenic effect, we hypothesized that PAI-1 may regulate lymphangiogenesis associated at least with metastatic dissemination of cancer cells. To address this issue, we studied the impact of PAI-1 deficiency in various murine models of tumoral lymphangiogenesis. Wild-type PAI-1 proficient mice were used as controls. We provide for the first time evidence that PAI-1 is dispensable for tumoral lymphangiogenesis associated with breast cancers either induced by mammary carcinoma cell injection or spontaneously appearing in transgenic mice expressing the polyomavirus middle T antigen (PymT) under the control of a mouse mammary tumor virus long-terminal repeat promoter (MMTV-LTR). We also investigated inflammation-related lymphatic vessel recruitment by using two inflammatory models. PAI-1 deficiency did neither affect the development of lymphangioma nor burn-induced corneal lymphangiogenesis. These novel data suggest that vascular remodelling associated with lymphangiogenesis and angiogenesis involve different molecular determinants. PAI-1 does not appear as a potential therapeutic target to counteract pathological lymphangiogenesis. [less ▲]

Age-related macular degeneration (AMD) is the leading cause of blindness in elderly patients. The more aggressive exudative form is characterized by abnormal blood-vessel development that occurs beneath ... [more ▼]

Age-related macular degeneration (AMD) is the leading cause of blindness in elderly patients. The more aggressive exudative form is characterized by abnormal blood-vessel development that occurs beneath the retina as a result of choroidal neovascularization (CNV). Vascular endothelial growth factor [VEGF) has emerged as the key mediator of CNV formation; this has led to intensive research on VEGF and the recent approval of anti-VEGF compounds by the US Food and Drug Administration. Despite this successful introduction of anti-angiogenic therapies into the clinical setting, there is still a lack of treatments that definitively reverse damaged vision. Here, we consider the importance of putative molecular targets other than VEGF that might have been underestimated. Emerging cellular mechanisms offer additional opportunities for innovative therapeutic approaches. [less ▲]

An adequate balance between serine proteases and their plasminogen activator inhibitor-1 (PAI-1) is critical for pathological angiogenesis. PAI-1 deficiency in mice is associated with impaired choroidal ... [more ▼]

An adequate balance between serine proteases and their plasminogen activator inhibitor-1 (PAI-1) is critical for pathological angiogenesis. PAI-1 deficiency in mice is associated with impaired choroidal neovascularization (CNV) and tumoral angiogenesis. In the present work, we demonstrate unexpected differences in the contribution of bone marrow (BM)-derived cells in these two processes regulated by PAI-1. PAI-1(-/-) mice grafted with BM-derived from wild-type mice were able to support laser-induced CNV formation but not skin carcinoma vascularization. Engraftment of irradiated wild-type mice with PAI-1(-/-) BM prevented CNV formation, demonstrating the crucial role of PAI-1 delivered by BM-derived cells. In contrast, the transient infiltration of tumor transplants by local PAI-1-producing host cells rather than by BM cells was sufficient to rescue tumor growth and angiogenesis in PAI-1-deficient mice. These data identify PAI-1 as a molecular determinant of a local permissive soil for tumor angiogenesis. Altogether, the present study demonstrates that different cellular mechanisms contribute to PAI-1-regulated tumoral and CNV. PAI-1 contributes to BM-dependent choroidal vascularization and to BM-independent tumor growth and angiogenesis. [less ▲]